1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, the present invention provides an improved reverse transcription method that allows the synthesis of long cDNA species.
2. Description of Related Art
A number of methods have been employed over the years for the synthesis of complementary DNA (cDNA). All of these methods utilize a reverse transcriptase (RT) for first strand synthesis and either a DNA polymerase or reverse transcriptase for second strand synthesis.
One of the first methods for isolating high quality cDNA was described by Efstratiadis et al. (1976). These investigators took advantage of the fact that, for reasons not completely understood, a small percentage of single-stranded cDNA will form hairpin structures at their 3xe2x80x2 ends. The hairpin structure could be used for priming of second strand synthesis and the hairpin was subsequently digested by S1 nuclease prior to cloning into a vector. Insertion into the vector was accomplished by using terminal transferase to form complementary homopolymeric tails at the ends of the vector and the cDNA. Despite the usefulness of this approach there were several drawbacks. It was not clear that all cDNAs formed hairpin structures and thus these libraries may not have been completely representational. Also, some degradation of the cDNA may result from the S1 nuclease digestion.
A major advance in the preparation of cDNA was the replacement synthesis method for second strand synthesis first introduced by Okayama and Berg (1982) and later modified by Gubler and Hofiman (1983). In this method, the second strand is synthesized by a nick translation procedure in which the mRNA strand is nicked by RNase H producing primers that can be utilized by E. coli polymerase I. This method is very efficient and eliminates the need for a S1 nuclease reaction. It remains the method of choice for second strand cDNA synthesis. For first strand synthesis, the enzymes primarily used have been either from the Moloney Murine Leukemia Virus (MMLV) or the avian myeloblastosis virus (AMV). The AMV RT was somewhat preferred because its optimum temperature was 42xc2x0 C. compared to 37xc2x0 C. for the MMLV enzyme. However, recently, the MMLV gene has been mutated in order to eliminate the endogenous RNase H activity, and this modified enzyme referred to as Superscript RT (Gibco-BRL), is superior for the production of full-length cDNAs.
A major impediment to the production of full-length cDNAs by existing techniques has been the occurrence of secondary structure in the mRNA. These and perhaps other naturally occurring pause sites inhibit the progression of the reverse transcriptases, and thus prevent the synthesis of full-length first strand cDNA. A number of methods, including the use of methylmercury hydroxide to denature the mRNA, have been used to remove the secondary structure during first strand synthesis. However, these methods have not proven to be completely satisfactory. Methylmercury hydroxide, for example, in addition to being highly toxic, inhibits RTs to some extent.
Another method for eliminating secondary structure in mRNA is to perform first strand synthesis at higher temperatures. However, this method also is flawed because the half lives of the MMLV and AMV enzymes at high temperatures are significantly reduced. Recently, however, RTs that are active at extremely high temperatures have been isolated. Unfortunately, such enzymes are not highly processive and therefore are not sufficient for the synthesis of full-length first strand cDNA.
An expression cloning approach that utilized an Epstein-Barr virus-based cloning vector capable of replicating extrachromasomally in human cells has been attempted to produce long cDNAs. The pEBS7 vector could be used for the efficient transformation and expression of cDNAs in human cells (Peterson and Legerski, 1991). Using a library prepared from mRNA derived from HeLa cells, the inventor""s group was able to initially clone the gene that complements the xeroderma pigmentosum group C (XPC) gene (approximately 4 kb) (Legerski and Peterson, 1992). In addition, the cloning of the Cockayne""s syndrome group A (CSA) gene (Tebbs et al., 1995), and a gene, XRCC3, that complements a Chinese hamster ovary (CHO) DNA repair mutant (Henning et al., 1995) also was achieved. Furthermore, two additional genes, XRCC2 and XRCC9, that complement CHO DNA repair mutants, have been cloned using the pEBS7 libraries.
Despite these successes, it remains apparent that very long cDNAs, above five or six kb, still were not well represented in these libraries. All of the genes discussed above were four kb or less in length. Attempts to clone longer gene sequences by this method have been unsuccessful. This defines a deficiency in the art in the production of full length cDNAs that has yet to be addressed.
In a particular embodiment, the present invention provides a method for the synthesis of cDNA comprising the steps of (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer, dNTPs, (b) incubating the reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a normal temperature range to allow first strand synthesis; (c) incubating the reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand; (d) adding the first strand to a reaction mixture for the synthesis of a second strand complementary to the first strand wherein the second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of the second strand and incubating the reaction mixture under conditions to allow the formation of a double-stranded cDNA. In specific embodiments, steps b and c are repeated. Steps b and c may be repeated once, twice, three, four or more times. More particularly, steps b and c are repeated until the appropriate length of first strand of the cDNA is generated.
In specific embodiments, the reaction mixture of step (a) further may comprise an RNase inhibitor. In other embodiments, the second strand synthesis reaction mixture of step (d) further comprises DEPC-treated H20. In still further embodiments, the second strand synthesis reaction mixture of step (d) further comprises RNase H. Certain embodiments further comprise the step of amplifying the double-stranded cDNA molecule of step (d). More particularly, the step of amplifying comprises PCR.
In specific embodiments, the temperature of step (b) is between about 37xc2x0 C. and about 43xc2x0 C. In other embodiments, the temperature of step (c) about 56xc2x0 C. and about 95xc2x0 C. The temperature in step (b) will be the temperature range optimal for any processive RT enzyme. The temperature range in step (c) will be any temperature range optimal for a thermostable RT. In specific examples, the processive reverse transcriptase may be selected from the group consisting of Superscript(trademark); AMV Reverse Transcriptase, M-MLV Reverse Transcriptase. In particular examples, the thermostable reverse transcriptase is selected from the group consisting of Retrotherm(trademark); Thermoscript(trademark) and Tth reverse transcriptase.
In other embodiments, it is envisioned that the DNA polymerase is thermostable or non-thermostable. The DNA polymerase may be selected from the group consisting of DNA Polymerase I, T4 DNA Polymerase, DNA Polymerase I Klenow fragment, PLATINUM taq(trademark). More particularly, the thermostable DNA polymerase may be selected from the group consisting of Tfl DNA Polymerase, Taq DNA Polymerase, Tli DNA Polymerase, Tth DNA Polymerase, Vent(trademark), Deepvent(trademark) and pfu.
In particularly defined embodiments, the sample comprises between about 0.1 and picograms and 10 micrograms of polyA RNA. Of course this is an exemplary range and other ranges of polyA RNA also are contemplated for example from about 1 picogram to about 1 microgram; 10 picograms to about 900 nanograms; 20 picograms to about 800 nanograms; 30 picograms to about 700 nanograms; 40 picograms to about 600 nanograms; 50 picograms to about 500 nanograms; 60 picograms to about 400 nanograms; 70 picograms to about 300 nanograms; 80 picograms to about 200 nanograms. It will be understood by those of skill in the art that virtually any amount of polyA RNA may be present in the sample. Also it is contemplated that the RNA may be total RNA extract from a tissue. In particular embodiments, it is contemplated that the poly(A)+RNA is from a tumor. In specific embodiments it is contemplated that the reaction mixture comprises between 1 and 108 copies of the poly(A)+RNA. Any number of copies between this range also is specifically contemplated.
In specific embodiments, the method may further comprise the step of adding linkers to the double stranded cDNA. More particularly, the linkers are added by blunt end ligation.
Also contemplated is a method of increasing the length of cDNAs in a cDNA library comprising the steps of (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer and dNTPs, (b) incubating the reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a normal temperature range to allow first strand synthesis; (c) incubating the reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand; (d) adding the first strand to a reaction mixture for the synthesis of a second strand complementary to the first strand wherein the second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of the second strand and incubating the reaction mixture under conditions to allow the formation of double-stranded cDNA, and (e)amplifying the double-stranded cDNA of step (d); wherein incubation at the temperatures in steps (c) inhibits the formation of secondary mRNA structures thereby resulting in cDNA species that are longer than in those produced in a normal temperature range.
Another embodiments contemplates a method for the production of full length cDNAs comprising the steps of (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer and dNTPs; (b) incubating the reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a normal temperature range to allow first strand synthesis; (c) incubating the reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand; (d) adding the first strand to a reaction mixture for the synthesis of a second strand complementary to the first strand wherein the second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of the second strand and incubating the reaction mixture under conditions to allow the formation of a double-stranded cDNA molecule, and (e) amplifying the double-stranded cDNA molecule of step (d) wherein the inhibition of secondary structure formation in step (b) allows the production of long cDNA moieties.
In specific embodiments, the cDNA moiety has a size of between about 0.5 kB and 20 kB. Of course this is an exemplary size range, the present invention is directed towards providing a method of making cDNA by reverse transcription such that the secondary structures in RNA transcripts do not interfere with the elongation of the first strand of cDNA. The cDNA may be 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb or larger.
In specific embodiments, the ADNA encodes a gene selected from the group consisting of XPC, CSA, XRCC3, XRCC2, XRCC9, ATM, ATR, RAD3), DNA-PK, ERCC1, XPA, XPB, XPC, XPD, XPF, XPQ, CSB and HHR23B. In other embodiments, the cDNA encodes a gene related to colorectal carcinoma. More particularly, the colorectal carcinoma is hereditary colorectal carcinoma. In other embodiments, the colorectal carcinoma is sporadic colorectal carcinoma. In those embodiments in which the cancer is a hereditary colorectal carcinoma the gene may be selected from the group consisting of hMSH2, hMLH1, hPMS1, hPMS2 and GTBP. In those embodiments in which the colorectal carcinoma is sporadic colorectal carcinoma the gene may be selected from the group consisting of transforming growth factor b type II receptor, insulin-like growth factor II receptor, BAX and xcex22-microglobulin.
Also provided herein is a method for synthesizing long cDNA moieties comprising the steps of (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer and dNTPs, (b) incubating the reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a normal temperature range to allow first strand synthesis; (c) incubating the reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand; (d) adding the first strand to a reaction mixture for the synthesis of a second strand complementary to the first strand wherein the second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of the second strand and incubating the reaction mixture under conditions to allow the formation of a double-stranded cDNA, and (e) amplifying the double-stranded cDNA molecule of step (d); wherein the inhibition of secondary structure formation in step (b) allows the production of cDNA moieties that are longer than those obtained when such secondary structure formation is not inhibited.
Another embodiment provides a method for producing a library of cDNA species from a tumor comprising the steps of (a) providing a reaction mixture comprising a poly (A)+RNA extracted from the tumor, an oligonucleotide primer and dNTPs; (b) incubating the reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a normal temperature range to allow first strand synthesis; (c) incubating the reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand; (d) adding the first strand to a reaction mixture for the synthesis of a second strand complementary to the first strand wherein the second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of the second strand and incubating the reaction mixture under conditions to allow the formation of a double-stranded cDNA, and (e) amplifying the double-stranded cDNA molecule of step (d) and (f) inserting the cDNA into an appropriate vector. In specific embodiments, the tumor is a colorectal tumor.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.